Simultaneous enantioselective determination of triazole fungicide difenoconazole and its main chiral metabolite in vegetables and soil by normal-phase high-performance liquid chromatography

Herein is reported, for the first time, a simple and highly sensitive chiral high-performance liquid chromatography (HPLC) method for the simultaneous quantitative determination of difenoconazole stereoisomers and their hydroxylated metabolite difenoconazole alcohol (CGA-205375) enantiomers in vegetables and soil matrix. The separation of difenoconazole and CGA-205375 including their simultaneous enantioseparation was studied using four different polysaccharide-type chiral stationary phases (CSPs) in combination with n-hexane-polar organic alcohols mobile phase. Chiralcel OJ consisting of 25?% of cellulose tris(4-methylbenzoate) coated on wide-pore polysaccharide silica gel exhibited higher resolving ability compared to cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD) as well as to its similar amylose derivative (Chiralpak AD) CSPs for this particular set of chiral analytes. Baseline separation and simultaneous enantioseparation of difenoconazole and its metabolite CGA-205375 could be achieved under optimized separation conditions. Based on the established HPLC method, enantioselective analysis method for this fungicide and its main chiral metabolite in vegetables and soil matrix were developed and validated. Parameters including the matrix effect, linearity, precision, accuracy, and stability were evaluated. Under the optimal conditions, the mean recoveries from cucumber, tomato, and soil matrix ranged from 81.65 to 94.52?%, with relative standard deviations in the range of 1.05-8.32?% for all stereoisomers. Coefficients of determination R (2)?≥?0.998 were achieved for each enantiomer in the cucumber, tomato and soil matrix calibration curves within the range of 0.5-50?μg mL(-1). The limits of quantification for all enantiomers in three matrices were all below 0.1?μg mL(-1). The methodology was successfully applied for simultaneous enantioselective analysis of difenoconazole stereoisomers and their metabolite in the real samples, indicating its efficacy in investigating the environmental stereochemistry of difenoconazole in food and environmental matrix.     

Enantioseparations on amylose tris(5-chloro-2-methylphenylcarbamate) in nano-liquid chromatography and capillary electrochromatography

Amylose tris(5-chloro-2-methylphenylcarbamate) was coated onto native and aminopropylsilanized silica in order to prepare chiral stationary phases (CSP) for enantioseparations using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC). The effect of the nature of silica, the particle size and pore diameter, the chiral selector loading onto silica, the mobile phase composition and pH, as well as separation variables such as a linear flow rate of the mobile phase, applied voltage in CEC, etc. on the separation of enantiomers was studied. It was found that CSPs based on amylose tris(5-chloro-2-methylphenylcarbamate) can be used for preparation of stable capillary columns for enantioseparations by nano-LC and CEC in combination with polar organic and aqueous–organic mobile phases. Higher peak efficiency was observed in CEC than in nano-LC.     

Chiral separation of beta-adrenergic antagonists, profen non-steroidal anti-inflammatory drugs and chlorophenoxypropionic acid herbicides using teicoplanin as the chiral selector in capillary liquid chromatography

Three groups of structurally diverse chiral compounds were used to study the interaction mechanism responsible for stereoselective recognition with teicoplanin as chiral selector in capillary liquid chromatography. Teicoplanin-based chiral stationary phase (CSP) was used. The effect of the variation of mobile phase composition on retention and enantioselective separation was studied. The mobile phase composition suitable for enantioresolution of the various chiral compounds differed according to the interaction forces needed for chiral recognition. Mobile phases with high buffer portion (70-90 vol.%) were preferred for separation of enantiomers of profen non-steroidal anti-inflammatory drugs and chlorophenoxypropionic acid herbicides that require hydrophobic interactions, inclusion and pi-pi interactions for stereoselective recognition with teicoplanin. Higher concentration triethylamine in the buffer (0.5-1.0%) increased resolution of these acids. On the other hand, H-bonding and electrostatic interactions are important in stereoselective interaction mechanism of beta-adrenergic antagonists with teicoplanin. These interaction types predominate in the reversed phase separation mode with high organic modifier content (95% methanol) and in polar organic mobile phases. For this reason beta-adrenergic antagonists were best enantioresolved in the polar organic mode. The mobile phase composed of methanol/acetic acid/triethylamine, 100/0.01/0.01 (v/v/v), provided enantioresolution values of all the studied beta-adrenergic antagonists in the range 1.1-1.9. Addition of teicoplanin to the mobile phase, which was suitable for enantioseparation of certain compounds on the CSP, was also investigated. This system was used to dispose of nonstereoselective interactions of analytes with silica gel support that often participate in the interaction with CSPs. Very low concentration of teicoplanin in the mobile phase (0.1 mM) resulted in enantioselective separation of 2,2- and 2,4-chlorophenoxypropionic acids.     

Separation of chiral furan derivatives by liquid chromatography using cyclodextrin-based chiral stationary phases

The enantiomeric separation of a set of 30 new chiral furan derivatives has been achieved on native and derivatized beta-cyclodextrin stationary phases using high performance liquid chromatography (HPLC). The hydroxypropyl-beta-cyclodextrin (Cyclobond RSP), the 2,3-dimethyl-beta-cyclodextrin (Cyclobond DM), and the acetyl-beta-cyclodextrin (Cyclobond AC) stationary phases are the most effective chiral stationary phases (CSPs) for the separation of these racemates in the reverse phase mode. No enantioseparations have been observed on the native beta-cyclodextrin chiral stationary phase (Cyclobond I 2000) and only a few separations have been attained on the S-naphthylethyl carbamate beta-cyclodextrin (Cyclobond SN) and 3,5-dimethylphenyl carbamate beta-cyclodextrin (Cyclobond DMP) chiral stationary phases in the reverse phase mode. The polar organic and the normal phase mode on these CSPs are not effective for separation of these compounds. The characteristics of the analytes, including steric bulk, hydrogen bonding ability, and geometry, play an important role in the chiral recognition process. The pH affects the enantioseparation of compounds with ionizable groups and the addition of 0.5% methyl tert-butyl ether to the mobile phase significantly enhances the separation efficiency for some highly retained compounds.     

Separation of chiral sulfoxides by liquid chromatography using macrocyclic glycopeptide chiral stationary phases

A set of 42 chiral compounds containing stereogenic sulfur was prepared. There were 31 chiral sulfoxide compounds, three tosylated sulfilimines and eight sulfinate esters. The separations were done using five different macrocyclic glycopeptide chiral stationary phases (CSPs), namely ristocetin A, teicoplanin, teicoplanin aglycone (TAG), vancomycin and vancomycin aglycone (VAG) and seven eluents, three normal-phase mobile phases, two reversed phases and two polar organic mobile phases. Altogether the macrocyclic glycopeptide CSPs were able to separate the whole set of the 34 sulfoxide enantiomers and tosylated derivatives. Five of the eight sulfinate esters were also separated. The teicoplanin and TAG CSPs were the most effective CSPs able to resolve 35 and 33 of the 42 compounds. The three other CSPs each were able to resolve more than 27 compounds. The normal-phase mode was the most effective followed by the reversed-phase mode with methanol-water mobile phases. Few of these compounds could be separated in the polar organic mode with 100% methanol mobile phases. Acetonitrile was also not a good solvent for the resolution of enantiomers of sulfur-containing compounds, neither in the reversed-phase nor in the polar organic mode. The structure of the chiral molecules was compared to the enantioselectivity factors obtained with the teicoplanin and TAG CSP. It is shown that the polarity, volume and shape of the sulfoxide substituents influence the solute enantioselectivity factor. Changing the oxidation state of the sulfur atom from sulfoxides to sulfinate esters is detrimental to the compound's enantioselectivity. The enantiomeric retention order on the teicoplanin and TAG CSPs was very consistent: the (S)-(+)-sulfoxide enantiomer was always the less retained enantiomer. In contrast, the (R)-(-)-enantiomer was less retained by the ristocetin A, vancomycin and vancomycin aglycone columns, showing the complementarity of these CSPs. The macrocyclic glycopeptide CSPs provided broad selectivity and effective separations of chiral sulfoxides.     

Development of dinitrophenylated cyclodextrin derivatives for enhanced enantiomeric separations by high-performance liquid chromatography

The synthesis and evaluation of new dinitrophenyl (DNP) substituted beta-cyclodextrin (beta-CD) chiral stationary phases (CSPs) for the enantioseparation of various classes of chiral analytes by HPLC are presented. The dinitrophenyl substituted beta-CD derivatives are synthesized and covalently bonded to functionalized 5 microm spherical porous silica gel. These are the first reported derivatized cyclodextrin which contains pi-electron deficient substituents (i.e., pi-acidic moieties). The column performance in terms of their ability to separate enantiomers is evaluated. A variety of different dinitro-substituted aryl groups are investigated and compared. The pH of the mobile phase buffers, the buffer composition, the number and position of the dinitro groups on the phenyl ring substituent, the degree of substitution, and the bonding strategy all greatly affect the performance of the CSPs. A large variety of racemic compounds have been separated successfully on these CSPs. The bonded dinitrophenyl-derivatized cyclodextrins are stable in all three mobile phase modes, namely, the reversed-phase, polar organic, and normal phase modes. No degradation in column performance was observed in any mode of operation even after more than 1000 injections. The analytical applicability of these types of CSPs for enantiomeric separations is discussed in detail.     

Enantioseparation of vesamicol and novel vesamicol analogs by high-performance liquid chromatography on different chiral stationary phases

High-performance liquid chromatography enantioseparation of vesamicol and six novel azaspirovesamicols (amino alcohols) was accomplished on different chiral stationary phases (CSPs) by using an optical rotation based chiral detector for identification of the resolved enantiomers. The Pirkle-type column Reprosil Chiral-NR was found to be most suitable for chiral resolution in normal phase (NP) mode; all compounds could be enantioseparated successfully. Also the cellulose-based column Reprosil Chiral-OM showed appropriate separation properties by using NP conditions. The amylose-type column Reprosil Chiral-AM-RP was most suitable for enantioseparation in reversed phase (RP) mode; five out of seven compounds were resolved. This CSP showed a considerably higher capability for chiral recognition of vesamicol derivatives in RP mode than the corresponding cellulose-based column Reprosil Chiral-OM-RP. Enantioseparation with the teicoplanin aglycone-based column Reprosil Chiral-AA was successful under polar ionic mobile phase conditions.     

万古霉素及其苯异氰酸酯衍生物手性固定相的制备与应用

采用"一锅法",以1,6-二异氰酸正己酯作间隔臂,制备了万古霉素及苯异氰酸酯衍生化的万古霉素手性固定相。对拉米夫定、拉米夫定的L-薄荷醇酯、酞胺哌啶酮和盐酸氟西汀进行了手性分离研究,在极性有机相模式下,研究了流动相甲醇中冰醋酸-三乙胺浓度和比例对手性分离的影响,观察到两种手性固定相具有不同的手性识别能力。在万古霉素手性固定相上4种溶质都获得了基线分离;在苯异氰酸酯衍生化的手性固定相上除盐酸氟西汀外也均获得基线分离。     

Enantiomeric separations of chiral polychlorinated biphenyls on three polysaccharide-type chiral stationary phases by supercritical fluid chromatography

Enantiomeric separations of 18 chiral polychlorinated biphenyls (PCBs) were investigated on three polysaccharide-type chiral stationary phases (CSPs; Sino-Chiral OJ, Chiralpak IB, and Chiralcel OD) by supercritical fluid chromatography (SFC). With these commonly used polysaccharide CSPs, 17 PCBs except PCB 135 (R(S) = 0.81) were well resolved (R(S) > 1.5) under appropriate mobile phases and temperatures. Using Sino-Chiral OJ, 14 PCBs could be baseline-separated, while only one and nine PCBs could be completely separated using Chiralpak IB and Chiralcel OD, respectively. The influence of column temperature was studied for the optimization of resolution, as well as for the type and percentage of organic modifier in the mobile phase. The resolution decreased as the temperature increased in the range of 26-40 °C in which the enantiomeric separations were an enthalpy-driven process. The addition of modifiers in the mobile phase decreased the resolution of the PCB enantiomers, but it clearly shortened their retention time. These separation results indicate that SFC is a promising chromatographic technique for chiral separation and enantiopure standard preparation.     

淀粉及纤维素三(3-三氟甲基苯基氨基甲酸酯)手性固定相的制备及其性能评价

为了扩展多糖类手性固定相的种类,制备了基于淀粉及纤维素三(3-三氟甲基苯基氨基甲酸酯)的涂敷型手性固定相,以正己烷-异丙醇混合液为流动相,对8种手性化合物进行了高效液相色谱拆分。研究表明: 虽然与应用最广泛的分别以淀粉及纤维素三(3,5-二甲基苯基氨基甲酸酯)为手性选择因子的商品化手性柱Chiralpak AD和Chiralcel OD相比,所制备的手性固定相的手性分离能力较低,但纤维素三(3-三氟甲基苯基氨基甲酸酯)手性固定相显示出特异的手性识别能力,一些手性化合物在此固定相上得到了比在Chiracel OD上更好的分离;所制备的手性固定相的手性识别能力随流动相中异丙醇含量的降低而变好,当流动相中正己烷与异丙醇的体积比为95:5时所制备的手性固定相显示出相对较高的手性识别能力;总体来说,淀粉三(3-三氟甲基苯基氨基甲酸酯)手性固定相的手性识别能力稍强于纤维素三(3-三氟甲基苯基氨基甲酸酯)手性固定相,同时两种手性固定相的手性识别能力具有一定的互补性。     

Chiral separation of lenalidomide by liquid chromatography on polysaccharide‐type stationary phases and by capillary electrophoresis using cyclodextrin selectors

Complementary techniques were applied for the investigation of the chiral recognition and enantiomeric resolution of lenalidomide using various cyclodextrins and polysaccharides as chiral selectors. The high‐performance liquid chromatography enantioseparation of the anticancer drug was achieved using polysaccharide‐type chiral stationary phases in polar organic mode. Elution order and absolute configuration were elucidated by combined circular dichroism spectroscopy and time‐dependent density functional theory calculations after the isolation of pure enantiomers. Chiral selector dependent and mobile‐phase dependent reversal of the enantiomer elution order was observed, and the nonracemic nature of the lenalidomide sample was also demonstrated. Eight anionic cyclodextrins were screened for their ability to discriminate between the uncharged enantiomers by using capillary electrophoresis. Only two derivatives presented chiral interactions, these cases being interpreted in terms of apparent stability constants and complex mobilities. The best results were delivered by sulfobutylether‐β‐cyclodextrin, where quasi‐equal stability constants were recorded and the enantiodiscrimination process was mainly driven by different mobilities of the transient diastereomeric complexes. The optimized high‐performance liquid chromatography (Chiralcel OJ column, pure ethanol with 0.6 mL/min flow rate, 40°C) and capillary electrophoresis methods (30 mM sulfobutylether‐β‐cyclodextrin, 30 mM phosphate pH 6.5, 12 kV applied voltage, 10°C) were validated for the determination of 0.1% (R)‐lenalidomide as a chiral impurity, which could be important if a racemic switch is achieved.     

Analytical and semipreparative high performance liquid chromatography enantioseparation of new substituted 1-thiocarbamoyl-3,5-diaryl-4,5-dihydro-(1H)-pyrazoles on polysaccharide-based chiral stationary phases in normal-phase, polar organic and reversed-phase conditions

The direct HPLC enantioseparation of five pairs of new chiral pyrazole derivatives on coated cellulose- and amylose-based chiral stationary phases (Chiralpak AD, Chiralcel OJ and Chiralcel OJ-RH) and new immobilised amylose-based Chiralpak IA CSP was performed. Very high enantioselectivity factor (alpha) values were achieved in polar organic and reversed-phase conditions by using OJ-RH as CSP. Chiralpak IA exhibited an excellent chiral resolving ability in normal-phase mode and it allowed the enantioseparation of analytes investigated with resolution factors (Rs) >20. Due to its bonded nature, it was successfully employed at analytical and semipreparative scale in combination with normal-phase eluents containing "non-standards" solvents such as acetone.     

Enantioseparations in non-aqueous capillary electrochromatography using polysaccharide type chiral stationary phases

Enantioseparations of chiral compounds with different structures were studied in non-aqueous capillary electrochromatography (NAQ CEC). Three different polysaccharide derivatives, cellulose tris(3,5-dimethylphenylcarbamate) (Chiralcel OD), amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak AD) and cellulose tris(4-methylbenzoate) (Chiralcel OJ) were used as chiral stationary phases (CSPs). Methanolic or ethanolic ammonium acetate solutions served as a mobile phase. The effect of the type of the CSP, the loading of the chiral selector on wide-pore aminopropyl derivatized silica gel and operational parameters such as apparent pH, applied voltage, etc. on the EOF and chromatographic characteristics (alpha, N, Rs) were studied. NAQ CEC represents a valuable alternative and an extension to chiral separations by HPLC with common-size columns as well as to capillary LC and CEC in aqueous buffers.     

Novel strong cation-exchange type chiral stationary phase for the enantiomer separation of chiral amines by high-performance liquid chromatography

The preparation of novel brush-type chiral cation-exchange materials based on de novo designed synthetic low molecular mass selectors (SOs) and their evaluation for enantioselective separation of chiral amines by HPLC are presented. The SO as the functional unit for enantioselectivity contains a beta-aminocyclohexanesulfonic acid moiety and is readily accessible via straightforward synthesis in both enantiomeric forms yielding chiral stationary phases (CSPs) with opposite configurations, CSPs 1 and 2, and reversed elution orders. For the evaluation of these novel CSPs by HPLC a sound set of chiral amines, mainly amino-alcohol type drug molecules, was selected. The chromatographic evaluations were carried out using polar organic mobile phase conditions. All of the analytes could be baseline separated, compared to common CSPs in parts with excellent peak efficiencies (up to 70000 theoretical plates per meter for the second eluted enantiomer). A number of experimental parameters have been varied to look at and prove the underlying ion-exchange process on CSPs 1 and 2, and to reveal suitable conditions for their operation. In this context, the influence of proton activity in the mobile phase and the effects of varying concentration and type of the counterion as well as type of co-ion and of bulk solvent components were thoroughly investigated.     

Enantiomeric resolution of new aromatase inhibitors by liquid chromatography on cellulose chiral stationary phases

Analytical HPLC methods using derivatized cellulose chiral stationary phases were developed for the direct enantioseparation of substituted [1-(imidazo-1-yl)-1-phenylmethyl)]-benzothiazolinone and benzoxazolinone derivatives with one chiral center. Those analogues of fadrozole constitute new potent nonsteroidal inhibitors of aromatase (P450 arom). The separations were made using normal phase methodology with a mobile phase consisting of n-hexane-alcohol (ethanol, 1-propanol, or 2-propanol) in various proportions, and a silica-based cellulose tris-3,5-dimethylphenylcarbamate (Chiralcel OD-H), or tris-methylbenzoate (Chiralcel OJ). The effects of concentration of various aliphatic alcohols in the mobile phase were studied. A better separation was achieved on cellulose carbamate phase compared with the cellulose ester phase. The effects of structural features of the solutes along with the temperature of the column on the discrimination between the enantiomers were examined. Baseline separation (Rs > 1.5) was easily obtained in many cases.     

Synthesis and application of mono-6-(3-methylimidazolium)-6-deoxyperphenylcarbamoyl-beta-cyclodextrin chloride as chiral stationary phases for high-performance liquid chromatography and supercritical fluid chromatography

The synthesis of mono-6-(3-methylimidazolium)-6-deoxyperphenylcarbamoyl-beta-cyclodextrin chloride (MPCCD) and its application in chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) are being reported. This chiral selector is coated onto silica gel in different weight percentages (15, 20 and 35%, w/w) to obtain CSPs having different loading content. These new chiral stationary phases are tested using normal-phase HPLC for enantioseparation of racemic aromatic alcohols. Indeed, the enantiodiscrimination abilities of these CSPs are found to be influenced by the loading content of the chiral selector. Among the three columns (MPCCD-C15, MPCCD-C20 and MPCCD-C35), the best enantioseparation results are obtained using a column containing 20% (w/w) of MPCCD (MPCCD-C20). The resolution (R(s)) obtained for p-fluorophenylethanol, p-chlorophenylethanol, p-bromophenylethanol, p-iodophenylethanol and p-fluorophenyl-3-buten-1-ol using MPCCD-C20 ranges from 3.83 to 5.65. Good enantioseparation results are obtained for these analytes under SFC separation conditions using the MPCCD-C20 column.     

高效液相色谱使用两种类型的纤维素-三(对甲基苯甲酸酯)固定相手性拆分非洛地平的比较

以高效液相色谱手性固定相法对非洛地平(FEL)进行手性拆分。分别采用两种类型的纤维素-三(对甲基苯甲酸酯)手性柱Chiralcel OJ-R和Chiralcel OJ-H进行比较实验,以正己烷-异丙醇(90:10, v/v)为流动相,考察了流动相、柱温对保留及手性拆分的影响。实验显示,两柱拆分FEL的van’t Hoff图均发生了转折,在高温区域为焓驱动,在低温区域为熵驱动。两柱在温度升高时拆分FEL的分离度均提高,其中OJ-H的分离度优于OJ-R。两种手性柱对FEL具有相似的拆分机理。     

Chromatographic Enantioseparation of Ten Amino Acid Amide Derivatives on Three Polysaccharide-Based Chiral Stationary Phases

Enantioseparation of ten kinds of amino acid amide derivatives bearing aniline moieties on three polysaccharide-based chiral stationary phases (CSPs) was first systematically investigated. The chromatographic experiments were performed in the normal phase mode, namely, with n-hexane and 2-propanol as mobile phase. The effects of chiral columns, concentration of 2-propanol and column temperature on the enantioseparation were studied in detail. These compounds can be well resolved on Chiralcel OD-H column with the resolution above 1.5. Enantioseparation mechanism of chiral analytes and the CSPs are proposed based on the thermodynamic analysis of the experimental data. Our study establishes a simple, fast and efficient analytical method for amino acid amide derivatives by chiral HPLC, and provides a reference for enantioseparation of chiral amino acid amide derivatives and similar chiral compounds.     

Analytical and semipreparative resolution of ranolazine enantiomers by liquid chromatography using polysaccharide chiral stationary phases

Novel HPLC methods were developed for the analytical and semipreparative resolution of new antianginal drug ranolazine enantiomers. Good baseline enantioseparation was achieved using cellulose tris (3,5-dimethylphenylcarbamate) (CDMPC) chiral stationary phases (CSPs) under both normal-phase and polar organic modes. The validation of the analytical methods including linearity, LODs, recovery, and precision, and the semipreparative resolution of ranolazine racemate were carried out using methanol as mobile phase without any basic and acidic additives under polar organic mode, using CDMPC CSPs. At analytical scale, the elution times of both enantiomers were less than 7.5 min at 20 degrees C and 1.0 mL/min, with the separation factor (a) 1.88 and the resolution factor (R(s)) 2.95. At semipreparative scale, about 14.3 mg/h enantiomers could be isolated and elution times of both enantiomers were less than 13 min at 2.0 mL/min. To increase the throughput, the technique of overlapping injections was used. The first eluted enantiomer was isolated with a purity of 99.6% enantiomer excess (e.e.) and > 99.0% yield. The second enantiomer was isolated with a purity of 98.8% e.e. and > 99.0% yield. In addition, optical rotation and circular dichroism spectroscopy of both ranolazine enantiomers isolated were also investigated.